Molecular and General Genetics MGG

, Volume 222, Issue 2–3, pp 278–283 | Cite as

Structure of the beta- 1,3-1,4-glucanase gene ofBacillus macerans: Homologies to other beta-glucanases

  • Rainer Borriss
  • Knut Buettner
  • Pekka Maentsaelae


The nucleotide sequence of an 852 base pair (bp) DNA fragment containing the entire gene coding for thermostable beta- 1,3-1,4-glucanase ofBacillus macerans has been determined. ThebglM gene comprises an open reading frame (ORF) of 711 by (237 codons) starting with ATG at position 93 and extending to the translational stop codon TAA at position 804. The deduced amino acid sequence of the mature protein shows 70% homology to published sequences of mesophilic beta- 1,3-1,4-glucanases fromB. subtilis andB. amyloliquefaciens. The sequence coding for mature beta-glucanase is preceded by a putative signal peptide of 25 amino acid residues, and a sequence resembling a ribosome-binding site (GGAGG) before the initiation codon. By contrast with the processed protein, the N-terminal amino acid sequence constituting the putative leader peptide bears no or only weak homology to signal peptides of mesophilicBacillus endo-beta-glucanases. TheB. macerans signal peptide appears to be functional in exporting the enzyme to the periplasm inE. coli. More than 50% of the whole glucanase activity was localized in the periplasmic space and in the supernatant. Whereas homology to endo-1,4-beta-glucanases is completely lacking, a weak amino acid homology between the sequence surrounding the active site of phage T4 lysozyme and a sequence spanning residues 126 through 161 ofB. macerans endo-beta-glucanase could be identified.

Key words

Endo-1,3-1,4-beta-glucanase DNA sequence Bacillus macerans Active site T4 lysozyme 


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  1. Anderson MA, Stone BA (1975) A new substrate for investigating the specifity of beta-glucan hydrolases. FEBS Lett 52:202–207Google Scholar
  2. Borriss R (1981) Purification and characterization of an extracellular beta-glucanase fromBacillus IMET B376. Z Allg Mikrobiol 21:7–17Google Scholar
  3. Borriss R, Schroeder KL (1981) Beta- 1,3-1,4-glucanase in sporeforming microorganisms. V The efficiency of beta-glucanase in reducing the viscosity of wort. Zentralbl Bakteriol 136:330–340Google Scholar
  4. Borriss R, Zemek J, Augustin J, Pacova Z, Kuniak L (1980) Beta1,3-1,4-glucanase in spore-forming microorganisms. II. Production of beta-glucan hydrolases by variousBacillus species. Zentralbl Bakteriol 135:435–442Google Scholar
  5. Borriss R, Bäumlein H, Hofemeister J (1985) Expression inEscherichia coli of a cloned beta-glucanase gene fromBacillus amyloliquefaciens. Appl Microbiol Biotechnol 22:63–71Google Scholar
  6. Borriss R, Manteuffel R, Hofemeister J (1988) Molecular cloning of a gene coding for thermostable beta-glucanase fromBacillus macerans. J Basic Microbiol 28:3–10Google Scholar
  7. Bradford MM (1976) A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248–254Google Scholar
  8. Briggs MS, Gierasch LM (1986) Molecular mechanism of protein secretion. The role of the signal sequence. Adv Protein Chem 38:109–180Google Scholar
  9. Cantwell BA, McConnell DJ (1983) Molecular cloning and expression ofBacillus subtilis beta-glucanase gene inEscherichia coli. Gene 23:211–219Google Scholar
  10. Clarke J, Yaguchi M (1985) The role of carboxyl groups in the function of endo-beta-1,4-glucanase fromSchizophyllum commune. Fur J Biochem 149:233–238Google Scholar
  11. Cornelis P, Digneffe C, Willemot K (1982) Cloning and expression of aBacillus coagulans amylase gene inEscherichia coli. Mol Gen Genet 186:507–511Google Scholar
  12. Erfle JD, Teather RM, Wood PJ, Irvin, JE (1988) Purification and properties of a 1,3-1,4-beta-d-glucanase (lichenase, 1,31,4-beta-d-glucan- 4-glucanohydrolase, E.C. fromBacterioides succinogenes cloned inEscherichia coli. Biochem J 255:833–841Google Scholar
  13. Fincher GB, Lock PA, Morgan MM, Lingelbach K, Wettenhall REH, Mercer JFB, Brandt A, Thomson, KK (1986) Primary structure of the (1–3,1–4)-beta-d-glucan 4-glucanohydrolase from barley aleurone. Proc Nail Acad Sci USA 83:2081–2085Google Scholar
  14. Fukumori F, Sasihara N, Kudo T, Horikoshi K (1986) Nucleotide se-sequences of two cellulase genes from alkalophilicBacillus sp. strain N4 and their strong homology. J Bacteriol 168:479–485Google Scholar
  15. Fukumori F, Kudo T, Sasihara N, Nagata Y, Ito K, Horikoshi K (1989) The third cellulase of alkalophilicBacillus sp. strain N-4: evolutionary relationships within thecel gene family. Gene 76:289–298Google Scholar
  16. Graebnitz F, Ruecknagel KP, Seil M, Staudenbauer WL (1989) Nucleotide sequence of theClostridium thermocellum bg1B gene encoding thermostable beta-glucosidase B: homology to fungal beta-glucosidases. Mol Gen Genet 217:70–76Google Scholar
  17. Hanahan D (1985) Techniques for transformation ofE. coli. In: DM Glover (ed) DNA cloning, a practical approach, vol 1. IRL Press, Oxford, pp 109–135Google Scholar
  18. Hofemeister J, Kurtz A, Borriss R, Knowles J (1986) The betaglucanase gene fromBacillus amyloliquefaciens shows extensive homology with that ofBacillus subtilis. Gene 49:177–187Google Scholar
  19. Hoj PB, Rodriguez EB, Stick RV, Stone BA (1989) Differences in active site structure in a family of beta-glucan endohydrolases deduced from the kinetics of inactivation by epoxyalkyl betaoligoglucosides. J Biol Chem 264:4939–4947Google Scholar
  20. Imanaka T, Shihazaki M, Takagi M (1986) A new way of enhancing the thermostability of proteases. Nature 324:695–697Google Scholar
  21. Kramer W, Drutsa V, Jansen HW, Kramer B, Pflugfelder M, Fritz HJ (1984) The gapped duplex DNA approach to oligonucleotide-directed mutation construction. Nucleic Acids Res 12:9441–9456Google Scholar
  22. Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132Google Scholar
  23. Legler G, Bause E (1973) Epoxyalkyl oligo (1–4) beta-d-glucosides as active-site directed inhibitors of cellulases. Carbohydr Res 28:45–52Google Scholar
  24. Mackay RM, Lo A, Willick G, Zuker M, Baird S, Dove M, Moranelli F, Seligy V (1986) Structure of aBacillus subtilis endo-beta1,4-glucanase gene. Nucleic Acid Res 14:9159–9170Google Scholar
  25. McLaughlin JR, Murray CL, Rabinowitz JC (1981) Unique features in the ribosome binding site sequence of the gram-positiveStaphylococcus aureus beta-lactamase gene. J Biol Chem 256:11283–11291Google Scholar
  26. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  27. Moran Jr CP, Lang N, LeGrice SFJ, Lee G, Stephens M (1982) Nucleotide sequences that signal the initiation of transcription and translation inBacillus subtilis. Mol Gen Genet 186:339–346Google Scholar
  28. Ohmann E, Rindt K-P, Borriss R (1969) Glucose-6-phosphat-dehydrogenase in autotrophen Mikroorganismen. Z Allg Mikrobiol 9:557–564Google Scholar
  29. Perret, JC (1954) Iodometric assay of penicillinase. Nature 174:1012–1013Google Scholar
  30. Robson LM, Chambliss GH (1987) Endo-beta-1,4-glucanase ofBacillus subtilis DLG. J Bacteriol 169:2017–2025Google Scholar
  31. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467Google Scholar
  32. Svenśon B (1988) Regional distant sequence homology between amylases, alpha-glucosidases and transglucanosylases. FEBS Lett 230:72–76Google Scholar
  33. Teeri TT, Lehtovaara P, Kauppinen S, Saluvuori J, Knowles J (1987) Homologous domains inTrichoderma reesei cellulolytic enzymes: gene sequence and expression of cellobiohydrolase. II. Gene 51:43–52Google Scholar
  34. Vernon CA, Banks B (1963) The enzymatic hydrolysis of glycosides. Proc Biochem Soc 86:7Google Scholar
  35. Weaver LH, Matthews BW (1987) Structure of bacteriophage T4 lysozyme refined at 1.7 Å resolution. J Mol Biol 193:189–199Google Scholar
  36. Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119Google Scholar
  37. Zappe H, Jones WA, Jones DT, Woods DR (1988) Structure of an endo-beta-1,4-glucanase gene fromClostridium acetobutylicum p262 showing homology with endoglucanase genes fromBacillus spp. Appl Envir Microbiol 54:1289–1292Google Scholar

Copyright information

© Springer-Verlag 1990

Authors and Affiliations

  • Rainer Borriss
    • 1
  • Knut Buettner
    • 2
  • Pekka Maentsaelae
    • 3
  1. 1.Zentralinstitut für Genetik und Kulturpflanzenforschung der Akademie der Wissenschaften der DDRGaterslebenGerman
  2. 2.Bereich Mikrobiologie, Sektion BiologicE.-M.-Arndt Universität GreifswaldGreifswaldGerman
  3. 3.Department of BiochemistryUniversity of TurkuTurkuFinland

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